Pharmacological inhibition of RAS overcomes FLT3 inhibitor resistance in FLT3-ITD+ AML through AP-1 and RUNX1

Summary AML is characterized by mutations in genes associated with growth regulation such as internal tandem duplications (ITD) in the receptor kinase FLT3. Inhibitors targeting FLT3 (FLT3i) are being used to treat patients with FLT3-ITD+ but most relapse and become resistant. To elucidate the resistance mechanism, we compared the gene regulatory networks (GRNs) of leukemic cells from patients before and after relapse, which revealed that the GRNs of drug-responsive patients were altered by rewiring their AP-1-RUNX1 axis. Moreover, FLT3i induces the upregulation of signaling genes, and we show that multiple cytokines, including interleukin-3 (IL-3), can overcome FLT3 inhibition and send cells back into cycle. FLT3i leads to loss of AP-1 and RUNX1 chromatin binding, which is counteracted by IL-3. However, cytokine-mediated drug resistance can be overcome by a pan-RAS inhibitor. We show that cytokines instruct AML growth via the transcriptional regulators AP-1 and RUNX1 and that pan-RAS drugs bypass this barrier.


Figure S1 :
Figure S1: Related to Figure 1.RNA-seq analysis of FLT3 inhibitor treated pa�ents A: Gels showing results of a PCR assays with FLT3-ITD primers from genomic DNA (gDNA) of pa�ents used in analysis showing that FLT3 ITD is present in samples before (1) and a�er (2) relapse from FLT3i.

Figure S2 :
Figure S2: Related to Figure 2. RUNX1 and AP-1 co-operate to deregulate genes a�er FLT3 inhibi�on A: KEGG pathway analysis of AP-1 target genes upregulated a�er FLT3i relapse in 2 or more pa�ents.B,C: Volcano plots of RNA-seq data from MOLM14 (B) and MV4-11 (C) cells treated with 10 nM Gilteri�nib.D,E: Venn diagrams showing overlap of upregulated (D) and downregulated (E) genes in MV4-11 and MOLM14 a�er FLT3i.F: KEGG pathway analyses of genes upregulated (le�) or downregulated (right) in both FLT3-ITD cell lines a�er Gilteri�nib treatment.G,H: Venn diagrams showing the overlap of FOS ChIP target genes and upregulated (G) or downregulated (H) genes in MV4-

Figure S3 :
Figure S3: Related to Figure 3. AP-1 and RUNX1 targets are deregulated a�er FLT3 inhibitor treatment.A: Histograms from flow cytometry analysis of the indicated cytokine surface receptors on primary cell samples collected before (grey) and a�er (blue) treatment with FLT3 inhibitors.B: Heatmap of the

Figure S4 :
Figure S4: related to Figure 3. RUNX1 is crucial to FLT3-ITD AML survival and shares a pathway with FLT3.A: Dose response curves of MV4-11 cells treated with Gilteri�nib (le�) CBFβ inhibitor (right) and ITD15 FLT3i relapse sample primary cells treated with CBFβ inhibitor All IC50 values show the mean IC50 ±

Figure S5 :
Figure S5: Related to Figure 4. Cytokines induce FLT3 inhibitor resistance in FLT3-ITD+ AML.A: Table of IC50 from primary cell samples treated with Gilteri�nib with cumula�ve addi�on of cytokines.Mean IC50 are shown (n=3) +/-standard devia�on.The condi�ons where a log2 increase in

Figure S6 :
Figure S6: Related to Figure 5. IL-3 suppresses genomic and transcriptomic changes which occur a�er FLT3 inhibiton in FLT3-ITD AML A: Average profile of ATAC peaks gained and lost in MOLM14 cells a�er Gilteri�nib treatment.B: Average profile of RUNX1 ChIP binding in MOLM14 cells a�er Gilteri�nib treatment.C: Average profile of FOS ChIP binding centred on RUNX1 ChIP peaks in the presence or absence of Gilteri�nib.D: Histograms of qPCR analysis of mRNA from MOLM14 cells treated with Gilteri�nib +/-10 ng/ml IL-3.Error bars show standard devia�ons and p-values calculated using Student's t-test are shown in the table below.E: Venn diagrams showing the overlap of 2-fold upregulated genes a�er Gilteri�nib treatment compared to untreated, and 2 fold downregulated genes in Gilteri�nib treated cells with IL-3 (above) and 2 fold downregulated genes a�er Gilteri�nib treatment compared to untreated, and 2fold upregulated genes in Gilteri�nib treated cells with IL-3 (above) in MOLM14 cells.F: Gene set enrichment analysis (GSEA) of genes downregulated (above) or upregulated (below) in Gilteri�nib treated MOLM14 cells ranked by the fold change between Gilteri�nib treated samples with and without IL-3.G: KEGG pathway analysis of genes deregulated in MOLM14 in Gilteri�nib treated cells compared to untreated and Gilteri�nib treated cells in the presence and absence of IL-3.H,I: GSEA (H) of cell cycle genes in untreated cells vs Gilteri�nib treated cells (above) and Gilteri�nib treated cells in the presence and absence of IL-3 (below), with box and whisker plot (I) showing rela�ve expression of cell cycle genes in the three treatments in MOLM14.J: GSEAs in MOLM14 cells treated with Gilteri�nib in the presence or absence of IL-3.GSEAs show upregulated genes which are RUNX1 ChIP targets (le�) or AP-1 targets (centre le�) and downregulated genes which are RUNX1 ChIP targets (centre right) or AP-1 targets (right) in MOLM14 a�er FLT3 inhibi�on.

Figure S7 :
Figure S7: Related to Figure 6.Pharmacological targe�ng of the RAS protein family is not vulnerable to cytokine mediated resistance